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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
72 h
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
(8R,9S,10R,13S,14S,17R)-17-ethynyl-10,13-dimethyl-1,2,4,7,8,9,10,11,12,13,14,15,16,17,-tetradecahydrospiro[cyclopenta[a]phenanthrene-3,2´-[1,3]dioxolan]-17-ol
EC Number:
610-527-3
Cas Number:
50407-76-6
Molecular formula:
C23H32O3
IUPAC Name:
(8R,9S,10R,13S,14S,17R)-17-ethynyl-10,13-dimethyl-1,2,4,7,8,9,10,11,12,13,14,15,16,17,-tetradecahydrospiro[cyclopenta[a]phenanthrene-3,2´-[1,3]dioxolan]-17-ol

Sampling and analysis

Analytical monitoring:
no

Test solutions

Details on test solutions:
The test organism was grown and hold in OECD medium which was used also for preparing test item solutions.
The Ethisterone-ketal has a very low solubility in aquatic solutions so its concentration cannot be determined by HPLC from aquatic solutions directly.
The stock solution was prepared by measuring 200.15 mg of test item into 2 000 ml OECD growth medium. After one hour stirring the suspension was filtered through 0.2 μm WhatmannTM ME24 filter. The concentration of the filtrate (stock solution) was 0.119 mg/L.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Test species: Pseudokirchneriella subcapitata (Korshikov) F.Hindák (formerly known as Selenastrum capricornutum and Raphidocelis subcapitata)
Source: Culture Collection of Algae and Protozoa, Scottish Marine Institute (www.ccap.ac.uk)
Batch: CCAP 278/4
Date of arriving: 27 October 2017

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Test temperature:
The vessels were kept in incubator at 21 ± 2 °C with continuous illumination. The treatments of control and test vessels were identical.
pH:
At the beginning of the test the pH values were measured in the remaining test solution of the concurrent test vessels at each dilution level.
At the end of the test the pH values were measured in each test vessel. The pH of test solutions was not adjusted at the beginning of the test.
Nominal and measured concentrations:
The stock solution was prepared by measuring 200.15 mg of test item into 2 000 ml OECD growth medium. After one hour stirring the suspension was filtered through 0.2 μm WhatmannTM ME24 filter. The concentration of the filtrate (stock solution) was 0.119 mg/L.
The toxic property of test item was investigated at five concentrations which were prepared by different dilutions of stock solution of test item. OECD medium was used for dilution of stock solution.
Details on test conditions:
Preparation and breeding of inoculum culture:
The inoculum culture was prepared four days before starting the test and it was bred in incubator at 21 ± 2 °C. At the end of incubation the cell density of the inoculum culture was determined with a Bürker chamber. The cell concentration was 1 305 556 cell/ml
reparation of stock solution:
The preparation of stock and test solutions was based on the preliminary non GLP range finding test [9]. As the Range Finding Test was not performed in compliance with the GLP-Regulations it is excluded from the Statement of Compliance in the final report, but the raw data of this test are archived under the study code of present study. The stock solution was prepared by measuring 200.15 mg of test item into 2 000 ml OECD growth medium. After one hour stirring the suspension was filtered through 0.2 μm WhatmannTM ME24 filter. The concentration of the filtrate (stock solution) was 0.119 mg/L.
Preparation of test solutions
Five test solutions of different concentration were prepared by mixing appropriate volume of OECD medium and stock solution of the test item. The maximum separation factor between of test vessels was 1.5. The following table shows the preparation of test solutions. The volume of the prepared dilution levels was 300 ml except the control solution which was 600 ml. The alga inoculum culture was diluted 263.16 times so the initial alga cell concentration in every dilution and the control levels were 4 961 cells/ml.
Preparation of test vessels:
Three replicates were prepared at every test and six at the control level. The vessels contained 75 ml test media.
Test procedure:
The test vessels were closed with silicone stopper and kept in the Binder KBW 400 incubator. For the proper mass transfer of carbon dioxide glass tubes with filter were placed through silicone stoppers. The test was maintained under static conditions for a period of 72 hours. The temperature during the test remained in the 21 ± 2 °C range. The temperature data were harvested by Extech SD200 datalogger. The vessels were shaken and illuminated continuously in the incubator. The vessels were placed randomly into the incubator and were repositioned daily after sampling.

Results and discussion

Effect concentrations
Duration:
72 h
Dose descriptor:
EC50
Remarks on result:
not determinable
Details on results:
According to the test results Ethisterone-ketal has no toxic effect on alga growth rate even at its saturated concentration (0.119 mg/L).

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
According to the Council Regulation (EC) 440/2008 Ethisterone-ketal is not hazardous to the aquatic environment [72 h EC50 (for algae or other aquatic plants) > saturated concentration of Ethisterone-ketal (0.119 mg/L)]. As the reported results are based on calculated concentration data which are gained out of a non-validated UHPLC method, the toxicity results must be interpreted by precaution!